This research proposal focuses on mechanistic studies of four key enzymes in the Wood-Ljungdahl pathway: pyruvate ferredoxin oxidoreductase (PFOR) CO dehydrogenase/acetyI-CoA synthase (CODH/ACS) methyltransferase (MeTr) and the corrinoid iron-sulfur protein (CFeSP). Studies on this system have enriched the areas of microbiology, biochemistry, and metallobiochemistry in revealing the structures of macromolecular channels and previously unknown metal clusters. These studies are providing insight into how the proteins of the Wood-Ljungdahl pathway use bioorganometallic intermediates in catalyzing group transfer reactions and C-C and C-S bond formation. This system serves as a paradigm for understanding bioinorganic chemical principles, complex protein-protein interactions, and how proteins coordinate redox reactions with chemical catalysis. Studies on PFOR will define the electronic structure of a substrate-derived hydroxyethyl-thiamine pyrophosphate radical intermediate, elucidate a novel biochemical role for CoA in gating an electron transfer reaction between the radical and FeS clusters in the protein, and assess the proposed existence of a carbon dioxide channel between PFOR and CODH. Studies on CODH/ACS are aimed at optimizing active heterologous expression of the monofunctional (CODH, ACS) and bifunctional (CODH/ACS) enzymes, establishing catalytic and physiological role(s) of the NiNi and CuNi forms of ACS, characterizing the interactions between substrates and CODH/ACS and the order of substrate binding, and elucidating how CODH and ACS communicate and coordinate the supply of and demand for the toxic gas CO. Our planned studies on MeTr and the CFeSP are aimed at identifying how MeTr activates the methyl group of methyltetrahydrofolate by protonation at N5 to promote formation of the first organometallic intermediate in the Wood-Ljungdahl pathway, determining the crystal structure of the CFeSP and its complex with MeTr, and establishing how coordination chemistry involving the lower axial ligand of the CfeSP promotes catalysis and redox activation.